Methods and kits for isolating biological target materials using silica magnetic particles

ABSTRACT

The present invention provides methods for isolating biological target materials, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from other substances in a medium using silica magnetic particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. The preferred embodiments of magnetic silica particles used in the methods and kits of the present invention are capable of forming a complex with at least 2 μg of biological target material per milligram of particle, and of releasing at least 60% of the material from the complex in the elution step of the method. The methods of the present invention produce isolated biological target material which is substantially free of contaminants, such as metals or macromolecular substances, which can interfere with further processing or analysis, if present.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Reference is made to concurrently filed U.S. patent applicationSer. No. ______, entitled “Silica Adsorbent on Magnetic Substrate”,which application is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for separating orisolating a biological target material from other substances in a mediumto produce an isolated material of sufficient purity for furtherprocessing or analysis. The present invention particularly relates tomethods for separating or isolating biological target materials usingmagnetically responsive particles capable reversibly binding thematerial. The present invention more specifically relates to methods forseparating or isolating biological target materials using at least onemagnetically responsive particle comprising silica or a silicaderivative such as silica gel which reversibly binds the biologicaltarget material thereof.

BACKGROUND OF THE INVENTION

[0003] Many molecular biological techniques such as reversetranscription, cloning, restriction analysis, and sequencing involve theprocessing or analysis of biological materials. These techniquesgenerally require that such materials be substantially free ofcontaminants capable of interfering with such processing or analysisprocedures. Such contaminants generally include substances that block orinhibit chemical reactions, (e.g. nucleic acid or proteinhybridizations, enzymatically catalyzed reactions, and other types ofreactions, used in molecular biological techniques), substances thatcatalyze the degradation or depolymerization of a nucleic acid or otherbiological material of interest, or substances that provide “background”indicative of the presence in a sample of a quantity of a biologicaltarget material of interest when the nucleic acid is not, in factpresent in the sample. Contaminants also include macromolecularsubstances from the in vivo or in vitro medium from which a nucleic acidmaterial of interest is isolated, macromolecular substances such asenzymes, other types of proteins, polysaccharides, or polynucleotides,as well as lower molecular weight substances, such as lipids, lowmolecular weight enzyme inhibitors or oligonucleotides. Contaminants canalso be introduced into a target biological material from chemicals orother materials used to isolate the material from other substances.Common contaminants of this last type include trace metals, dyes, andorganic solvents.

[0004] Obtaining DNA or RNA sufficiently free of contaminants formolecular biological applications is complicated by the complex systemsin which the DNA or RNA is typically found. These systems, e.g., cellsfrom tissues, cells from body fluids such as blood, lymph, milk, urine,feces, semen, or the like, cells in culture, agarose or polyacrylamidegels, or solutions in which target nucleic acid amplification has beencarried out, typically include significant quantities of contaminantsfrom which the DNA or RNA of interest must be isolated before being usedin a molecular biological procedure.

[0005] Conventional protocols for obtaining DNA or RNA from cells aredescribed in the literature. See, e.g. Chapter 2 (DNA) and Chapter 4(RNA) of F. Ausubel et al., eds., Current Protocols in MolecularBiology, Wiley-Interscience, New York (1993). Conventional DNA isolationprotocols generally entail suspending the cells in a solution and usingenzymes and/or chemicals, gently to lyse the cells, thereby releasingthe DNA contained within the cells into the resulting lysate solution.For isolation of RNA, the conventional lysis and solubilizationprocedures include measures for inhibition of ribonucleases andcontaminants to be separated from the RNA including DNA.

[0006] Many conventional protocols in use today also generally entailuse of phenol or an organic solvent mixture containing phenol andchloroform to extract additional cellular material such as proteins andlipids from a conventional lysate solution produced as described above.The phenol/chloroform extraction step is generally followed byprecipitation of the nucleic acid material remaining in the extractedaqueous phase by adding ethanol to that aqueous phase. The precipitateis typically removed from the solution by centrifugation, and theresulting pellet of precipitate is allowed to dry before beingresuspended in water or a buffer solution for further processing oranalysis.

[0007] Conventional nucleic acid isolation procedures have significantdrawbacks. Among these drawbacks are the time required for the multipleprocessing steps necessary in the extractions and the dangers of usingphenol or chloroform. Phenol causes severe burns on contact. Chloroformis highly volatile, toxic and flammable. Those characteristics requirethat phenol be handled and phenol/chloroform extractions be carried outin a fume hood.

[0008] Another undesirable characteristic of phenol/chloroformextractions is that the oxidation products of phenol can damage nucleicacids. Only freshly redistilled phenol can be used effectively, andnucleic acids cannot be left in the presence of phenol. Generally also,multi-step procedures are required to isolate RNA afterphenol/chloroform extraction. Ethanol (or isopropanol) precipitationmust be employed to precipitate the DNA from aphenol/chloroform-extracted aqueous solution of DNA and remove residualphenol and chloroform from the DNA. Further, ethanol (or isopropanol)precipitation is required to remove some nucleoside triphosphate andshort (i.e., less than about 30 bases or base pairs) single or doublestranded oligonucleotide contaminants from the DNA. Moreover, under thebest circumstances such methods produce relatively low yields ofisolated nucleic acid material and/or isolated nucleic acid materialcontaminated with impurities.

[0009] There is a need recognized in the art for methods, that aresimpler, safer, or more effective than the traditional phenol/chloroformextraction/ethanol precipitation methods to isolate DNA and/or RNAsufficiently for manipulation using molecular biological procedures.

[0010] Fractionation of DNA recovered from cells according to size isrequired for many molecular biological procedures. Such fractionation istypically accomplished by agarose or polyacrylamide gel electrophoresis.For analysis or treatment by a molecular biological procedure afterfractionation, the DNA in the fraction(s) of interest must be separatedfrom contaminants, such as agarose, other polysaccharides,polyacrylamide, acrylamide, or acrylic acid, in the gel used in suchelectrophoresis. Thus, there is also a need in the art for methods toaccomplish such separations.

[0011] Methods for amplifying nucleic acids or segments thereof, such asthe well known polymerase chain reaction (PCR) process (see, e.g., U.S.Pat. No. 4,683,202), yield solutions of complex mixtures of enzymes,nucleoside triphosphates, oligonucleotides, and other nucleic acids.Typically, the methods are carried out to obtain an highly increasedquantity of a single nucleic acid segment (“target segment”). Often itis necessary to separate this target segment from other components inthe solution after the amplification process has been carried out. Thusthere is a further need in the art for simple methods to accomplishthese separations.

[0012] Silica materials, including glass particles, such as glasspowder, silica particles, and glass microfibers prepared by grindingglass fiber filter papers, and including diatomaceous earth, have beenemployed in combination with aqueous solutions of chaotropic salts toseparate DNA from other substances and render the DNA suitable for usein molecular biological procedures. See U.S. Pat. No. 5,075,430 andreferences cited therein, including Marko et al., Anal. Biochem. 121,382-387 (1982) and Vogelstein et al., Proc. Natl. Acad. Sci. (USA) 76,615-619 (1979). See also Boom et al., J. Clin. Microbiol. 28, 495-503(1990). With reference to intact glass fiber filters used in combinationwith aqueous solutions of a chaotropic agent to separate DNA from othersubstances, see Chen and Thomas, Anal. Biochem. 101, 339-341 (1980).Vogelstein et al., supra, suggest that silica gel is not suitable foruse in DNA separations. With regard to separation of RNA using silicamaterials and chaotropic agents, see Gillespie et al., U.S. Pat. No.5,155,018.

[0013] Glass particles, silica particles, silica gel, and mixtures ofthe above have been configured in various different forms to producematrices' capable of reversibly binding nucleic acid materials whenplaced in contact with a medium containing such materials in thepresence of chaotropic agents. Such matrices are designed to remainbound to the nucleic acid material while the matrix is exposed to anexternal force such as centrifugation or vacuum filtration to separatethe matrix and nucleic acid material bound thereto from the remainingmedia components. The nucleic acid material is then eluted from thematrix by exposing the matrix to an elution solution, such as water oran elution buffer. Numerous commercial sources offer silica-basedmatrices designed for use in centrifugation and/or filtration isolationsystems. See, e.g. Wizard™ DNA purification systems line of productsfrom Promega Corporation (Madison, Wis., U.S.A.); or the QiaPrep™ lineof DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.)

[0014] Magnetically responsive particles (hereinafter, “magneticparticles”) have conventionally been used to isolate and purifypolypeptide molecules such as proteins or antibodies. In recent years,however, magnetic particles and methods for using magnetic particleshave been developed for the isolation of nucleic acid materials. Severaldifferent types of magnetic particles designed for use in nucleic acidisolation are described in the literature, and many of those types ofparticles are available from commercial sources. Such magnetic particlesgenerally fall into either of two categories, those designed toreversibly bind nucleic acid materials directly, and those designed todo so through at least one intermediary substance. The intermediarysubstance is referred to herein as a “label.”

[0015] The magnetic particles designed to bind nucleic acid materialsindirectly are generally used to isolate a specific nucleic acidmaterial, such as mRNA, according to the following basic isolationprocedure. First, a medium containing a nucleic acid material is placedin contact with a label capable of binding to the nucleic acid materialof interest. For example, one such commonly employed label, biotinylatedoligonucleotide deoxythymidine (oligo-dT), forms hydrogen bonds with thepoly-adenosine tails of mRNA molecules in a medium. Each label soemployed is designed to bind with a magnetically responsive particle,when placed into contact with the particle under the proper bindingconditions. For example, the biotin end of a biotinylated oligo-dT/mRNAcomplex is capable of binding to streptavidin moieties on the surface ofa streptavidin coated magnetically responsive particle. Severaldifferent commercial sources are available for streptavidin magneticparticles and reagents designed to be used in mRNA isolation usingbiotinylated oligo-dT as described above. See, e.g. PolyATtract® Series9600™ mRNA Isolation System from Promega Corporation; or the ProActive™line of streptavidin coated microsphere particles from BangsLaboratories (Carmel, Ind., U.S.A.). Magnetic particles and labelsystems have also been developed which are capable of indirectly bindingand isolating other types of nucleic acids, such as double-stranded andsingle-stranded PCR templates. See, e.g. BioMag™ superparamagneticparticles from Advanced Magnetics, Inc. (Cambridge, Mass., U.S.A.)Indirect binding magnetic separation systems for nucleic acid isolationor separation all require at least three components, i.e. magneticparticles, a label, and a medium containing the nucleic acid material ofinterest. The label/nucleic acid binding reaction and label/particlebinding reaction often require different solution and/or temperaturereaction conditions from one another. Each additional component orsolution used in the nucleic acid isolation procedure adds to the riskof contamination of the isolated end product by nucleases, metals, andother deleterious substances.

[0016] A few types of magnetic particles have also been developed foruse in the direct binding and isolation of biological materials,particularly nucleic acid. One such particle type is a magneticallyresponsive glass bead, preferably of a controlled pore size. See, e.g.Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park,N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat.Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tends tobind so tightly to glass, however, that it can be difficult to removeonce bound thereto. Therefore, elution efficiencies from magnetic glassparticles tend to be low compared to elution efficiencies from particlescontaining lower amounts of a nucleic acid binding material such assilica.

[0017] A second type of magnetically responsive particles designed foruse in direct binding and isolation of biological materials,particularly nucleic acid, are particles comprised of agarose embeddedwith smaller ferromagnetic particles and coated with glass. See, e.g.U.S. Pat. No. 5,395,498. A third type of magnetically responsiveparticle, a particle capable of directly bind enzymes, proteins,hormones, or antibodies, is produced by incorporating magnetic materialsinto the matrix of polymeric silicon dioxide compounds. See, e.g. GermanPatent No. DE 43 07 262 A1. The latter two types of magnetic particles,the agarose particle and the polymeric silicon dioxide matrix, tend toleach iron into a medium under the conditions required to bindbiological materials directly to each such magnetic particle. It is alsodifficult to produce such particles with a sufficiently uniform andconcentrated magnetic capacity to ensure rapid and efficient isolationof nucleic acid materials bound thereto.

[0018] What is needed is a method for isolating biological entities,particularly nucleic acids, using a magnetically responsive particlecapable of rapidly and efficiently directly isolating such entitiessufficiently free of contaminants to be used in molecular biologyprocedures.

SUMMARY OF THE INVENTION

[0019] Briefly, in one aspect, the present invention comprises a methodof isolating a biological target material from other materials in amedium by:

[0020] providing a medium including the biological target material;

[0021] providing silica magnetic particles;

[0022] forming a complex of the silica magnetic particles and thebiological target material by combining the silica magnetic particlesand the medium;

[0023] removing the complex from the medium by application of anexternal magnetic field and

[0024] separating the biological target material from the complex byeluting the biological target material whereby the isolated biologicaltarget material is obtained.

[0025] In a further aspect, the present invention is a method ofisolating a biological target material of interest from other materialsin a medium using silica magnetic particles capable of reversiblybinding at least 2 micrograms of biological target material permilligram of silica magnetic particles, and of releasing at least 60% ofthe biological target material bound thereto. In preferred practices ofthe present method, at least about 4 micrograms of biological targetmaterial per milligram of silica magnetic particle is bound thereto andat least about 75% of the biological target material adhered to thesilica magnetic particles is subsequently eluted. The biological targetmaterial isolated according to the method of this invention ispreferably nucleic acid.

[0026] A preferred practice of the method of the present inventioncomprises the following steps. First, a mixture is formed comprising themedium and the silica magnetic particles. Second, the biological targetmaterial is adhered to the silica magnetic particles in the mixture.Third, the silica magnetic particles are removed from the mixture usingan external force, most preferably using a magnetic force, and Fourth,at least 60% of the biological target material adhered to the silicamagnetic particle is eluted by contacting the particle with an elutionsolution.

[0027] In another aspect, the present invention is a method of isolatingplasmid DNA from other materials in a medium using a preferred form ofsilica magnetic particle, i.e., siliceous-oxide coated magneticparticle, wherein the preferred particles are capable of binding atleast 2 micrograms of the plasmid DNA material per milligram ofparticle, and of releasing at least 60% of the plasmid DNA materialbound thereto. A preferred practice of the methods of this aspect of theinvention comprise the following steps. First, a mixture is formedcomprising the medium including plasmid DNA, the siliceous-oxide coatedmagnetic particle, and a chaotropic salt. Second, the plasmid DNA isadhered to the siliceous-oxide coated magnetic particle in the mixture.Third, the siliceous-oxide coated magnetic particle is removed from themixture using an external force, most preferably using a magnetic field.Fourth, at least 60% of the plasmid DNA adhered to the siliceous-oxidecoated magnetic particle is eluted by contacting the particle with anelution solution.

[0028] In a further aspect, the present invention is a kit for isolatinga biological target material from a medium containing the same, the kitcomprising an aliquot of siliceous-oxide coated magnetic particlessuspended in an aqueous solution in a first container, wherein theparticles have the capacity to reversibly bind at least 2 micrograms ofthe biological target material per milligram of particle. Optionally,the kit may include other components needed to isolate a biologicaltarget material from a medium containing the same according to themethods of the present invention.

[0029] As used herein, the term “magnetic particles” refers to materialswhich have no magnetic field but which form a magnetic dipole whenexposed to a magnetic field, i.e., materials capable of being magnetizedin the presence of a magnetic field but which are not themselvesmagnetic in the absence of such a field. The term “magnetic” as used inthis context includes materials which are paramagnetic orsuperparamagnetic materials. The term “magnetic”, as used herein, alsoencompasses temporarily magnetic materials, such as ferromagnetic orferrimagnetic materials with low Curie temperatures, provided that suchtemporarily magnetic materials are paramagnetic in the temperature rangeat which silica magnetic particles containing such materials are usedaccording to the present methods to isolate biological materials.

[0030] The term “silica magnetic particle” refers to a magnetic particlecomprised of silica in the form of silica gel, siliceous oxide, solidsilica such as glass or diatamaceous earth, or a mixture of two or moreof the above. The term “silica gel” as used herein refers tochromatography grade silica gel, a substance which is commerciallyavailable from a number of different sources. Silica gel is mostcommonly prepared by acidifying a solution containing silicate, e.g.sodium silicate, to a pH of less than 10 or 11 and then allowing theacidified solution to gel. See, e.g. silica preparation discussion inKurt-Othmer Encyclopedia of Chemical Technology, Vol. 6, 4th ed., MaryHowe-Grant, ed., John Wiley & Sons, pub., 1993 , pp. 773-775. The term“silica magnetic particle” as used herein preferably refers to particleswith the characteristics described above having the capacity to bind atleast 2 micrograms of biological target material per milligram of silicamagnetic particles and, independently, the capacity to release at least60% of the biological target material bound thereto in the elution stepof the present method. The silica magnetic particles used in the presentinvention preferably further comprise ferromagnetic materialincorporated into a silica gel matrix. The elution step in the isolationmethods of this invention are preferably accomplished withoutsubstantial contamination of the nucleic acid material by metal or metalcompounds (e.g., iron or iron compounds) or other objectionable speciesoriginating from the silica magnetic particles.

[0031] The term “glass particles” as used herein means particles ofcrystalline silicas (e.g., a-quartz, vitreous silica), even thoughcrystalline silicas are not formally “glasses” because they are notamorphous, or particles of glass made primarily of silica.

[0032] The term “siliceous-oxide coated magnetic particle” or “SOCMparticle” is used herein to refer to the most preferred form of silicamagnetic particle used in the methods and kits of the present invention.The SOCM particle is comprised of siliceous oxide coating a core of atleast one particle of superparamagnetic or paramagnetic material. TheSOCM particle used in the present method and kits also has an adsorptivesurface of hydrous siliceous oxide, a surface characterized by havingsilanol groups thereon. Target nucleic acid material, such as DNA orRNA, adhere to the adsorptive surface of the particle while othermaterial, particularly deleterious contaminants such as exonucleases, donot adhere to or co-elute from the particle with the nucleic acidmaterials. The physical characteristics of the SOCM particle and methodsfor producing such particles are disclosed in concurrently filed U.S.patent application Ser. No. ______, entitled “Silica Adsorbent onMagnetic Substrate,” the disclosure of which is incorporated byreference herein.

[0033] The present invention provides convenient and efficient means forisolating biological target material of interest from a variety ofdifferent media. A preferred aspect of the present method describedbriefly above, wherein magnetic force is used to remove the particlesfrom the media, offers significant advantages over conventionalisolation methods wherein a biological target material is reversiblybound to other silica material. Specifically, the magnetic removal stepof the method substitutes for vacuum filtration or centrifugation stepsrequired in conventional silica binding and elution isolation methods.It is, therefore, particularly amenable to being automated. Smalllaboratories or individual researchers frequently must purchasespecialized and expensive equipment to carry out such methods, such as avacuum manifold and vacuum for use in vacuum filtration or amicrocentrifuge for centrifugation methods. Contrastingly, magneticseparation of the present invention merely requires a concentratedmagnetic field such as is generated from a strong and readily availablemagnet. Inexpensive apparatuses specifically adapted for use inmolecular biology research context are also commercially available, suchas the MagneSphere® Technology Magnetic Separation Stand or thePolyATract® Series 9600™ Multi-Magnet (both available from PromegaCorporation, Madison, Wis., USA).

[0034] The biological target material isolated using the isolationmethod of the present invention is sufficiently free of contaminatingmaterial for additional processing or analysis using standard molecularbiology techniques. Applications of the present methods to isolatevarious different biological target materials from a variety ofdifferent media will become apparent from the detailed description ofthe invention below.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1 is a plot of the number of micrograms of plasmid DNA boundper microgram of plasmid DNA added to either magnetic controlled poreglass (CPG) particles or to silica magnetic particles.

[0036]FIG. 2 is a plot of the number of micrograms of plasmid DNA elutedfrom either magnetic CPG or silica magnetic particles versus the amountof plasmid DNA added to the particles prior to elution.

[0037]FIG. 3 is a plot of the binding data shown in FIG. 1 and theelution data shown in FIG. 2 obtained from magnetic CPG and silicamagnetic particles.

[0038]FIG. 4 is a fluorimage of an agarose gel stained with afluorescent dye, after fractionation of DNA fragments on the gel usinggel electrophoresis, wherein the DNA fragments were produced bydigesting lambda DNA with Hind III and by binding and eluting thefragments from silica magnetic particles.

[0039]FIG. 5 is a fluorimage of an agarose gel stained with afluorescent dye, after fractionation of DNA fragments on the gel usinggel electrophoresis, wherein the DNA fragments were produced bydigesting φX174 DNA with Hae III and by binding and eluting thefragments from silica magnetic particles.

[0040]FIG. 6 is a histogram plot of the number of counts per million(CPM) of ³²P-labeled RNA applied to, bound to, and released from silicamagnetic particles.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The biological target material isolated using the methods of thepresent invention is preferably a nucleic acid or a protein, morepreferably a nucleic acid material such as RNA, DNA, or a RNA/DNAhybrid. When the biological target material isolated using the presentmethods is a nucleic acid, it is preferably DNA, or RNA including butnot limited to plasmid DNA, DNA fragments produced from restrictionenzyme digestion, amplified DNA produced by an amplification reactionsuch as the polymerase chain reaction (PCR), single-stranded DNA, mRNA,or total RNA. The nucleic acid material isolated according to themethods of the present invention is even more preferably a plasmid DNAor total RNA.

[0042] Since nucleic acids are the most preferred biological targetmaterial isolated using the methods of the present invention, most ofthe detailed description of the invention below describes this preferredaspect of the present invention. However, the detailed description ofthis particular aspect of the present invention is not intended to limitthe scope of the invention. The present disclosure provides sufficientguidance to enable one of ordinary skill in the art of the presentinvention to use the methods of the present invention to isolatebiological target materials other than nucleic acid materials, e.g.,proteins or antibodies.

[0043] The present methods of isolating biological target material canbe practiced using any silica magnetic particle, but the methods arepreferably practiced using the SOCM form of silica magnetic particles.The present methods are also preferably practiced using silica magneticparticles with the following physical characteristics.

[0044] The silica magnetic particles used in the methods of thisinvention may be any one of a number of different sizes. Smaller silicamagnetic particles provide more surface area (one per weight unit basis)for adsorption, but smaller particles are limited in the amount ofmagnetic material which can be incorporated into such particles comparedto larger particles. The median particle size of the silica magneticparticles used in the present invention is preferably about 1 to 15 μm,more preferably about 3 to 10 μm, and most preferably about 4 to 7 μm.The particle size distribution may also be varied. However, a relativelynarrow monodal particle size distribution is preferred. The monodalparticle size distribution is preferably such that about 80% by weightof the particles are within a 10 μm range about the median particlesize, more preferably within an 8 μm range, and most preferably within a6 μm range.

[0045] The silica magnetic particle preferably used in the presentinvention has pores which are accessible from the exterior of theparticle. The pores are preferably of a controlled size rangesufficiently large to admit a biological target material, e.g., nucleicacid, into the interior of the particle and to bind to the silica gelmaterial on the interior surface of most such pores. The pores of themost preferred form of the silica magnetic particles are designed toprovide a large surface area of silica gel material capable of binding abiological target material, particularly nucleic material. The totalpore volume of a silica magnetic particle, as measured by nitrogen BETmethod, is preferably at least about 0.2 ml/g of particle mass. Of thetotal pore volume measured by nitrogen BET, preferably at least about50% of the pore volume is contained in pores having a diameter of 600 Åor greater.

[0046] The silica magnetic particles may contain substances, such astransition metals or volatile organics, which could adversely affect theutility of isolated biological target material substantiallycontaminated with such substances. Specifically, such contaminants couldaffect downstream processing, analysis, and/or use of the suchmaterials, for example, by inhibiting enzyme activity or nicking ordegrading the target material itself. Any such substances present in thesilica magnetic particles used in the present invention are preferablypresent in a form which does not readily leach out of the particle andinto the isolated biological target material produced according to themethods of the present invention. Iron is one such undesirablecontaminant, particularly when the biological target material is anucleic acid. Iron, in the form of magnetite, is present at the core ofa particularly preferred form of the silica magnetic particles of thepresent invention, i.e. the SOCM particles. Iron has a broad absorptionpeak between 260 and 270 nanometers (nm). Nucleic acids have a peakabsorption at about 260 nm, so iron contamination in a nucleic acidsample can adversely affect the accuracy of the results of quantitativespectrophotometric analysis of such samples. Any iron containing silicamagnetic particles used to isolate nucleic acids using the presentinvention preferably do not produce isolated nucleic acid materialsufficiently contaminated with iron for the iron to interfere withspectrophotometric analysis of the material at or around 260 nm.

[0047] The most prefered silica magnetic particles used in the methodsof the present invention, the SOCM particles, leach no more than 50 ppm,more preferably no more than 10 ppm, and most preferably no more than 5ppm of transition metals when assayed as follows. Specifically, 0.33 gof the particles (oven dried @10° C.) into 20 ml. of 1N HCI aqueoussolution (using deionized water). The resulting mixture is then agitatedonly to disperse the particles. After about 15 minutes total contacttime, a portion of the liquid from the mixture is then analyzed formetals content. Any conventional elemental analysis technique may beemployed to quantify the amount of transition metal in the resultingliquid, but inductively coupled plasma spectroscopy (ICP) is preferred.

[0048] Concurrently filed patent application Ser. No. ______, entitled“Silica Adsorbent on Magnetic Substrate” incorporated by referenceherein, discloses methods for producing SOCM particles suitable for usein the methods and kits of the present invention. The most preferredsuch method for producing SOCM particles for use in the presentinvention comprises the general steps of: (1) preparing magnetite coreparticles by aqueous precipitation of a mixture of FeCl₂ and FeCl₃. (2)depositing a siliceous oxide coating on the magnetite core particles byexposing a slurry of the particles to a mixture of SiO₂ and Na₂O for atleast about 45 minutes at a temperature of at least 60° C. and thenadding an acid solution to the mixture until the pH is lowered to a pHless than 9, (3) allowing the resulting slurry to age for at least about15 minutes, preferably while continuing to agitate the slurry, and (4)washing the particles. The deposition and aging steps of the preferredparticle production method described above can be repeated to producemultiple layers of siliceous oxide coating over the magnetite core, thusproviding additional insurance against leaching of metals from the coreinto the surrounding environment. SOCM particles produced by the methoddescribed above are most preferably treated by being subjected to a mildoxidizing step to further inhibit leaching from the core.

[0049] The biological target material isolated using the method of thepresent invention can be obtained from eukaryotic or prokaryotic cellsin culture or from cells taken or obtained from tissues, multicellularorganisms including animals and plants; body fluids such as blood,lymph, urine, feces, or semen; embryos or fetuses; food stuffs;cosmetics; or any other source of cells. Some biological targetmaterials, such as certain species of DNA or RNA are isolated accordingto the present method from the DNA or RNA of organelles, viruses,phages, plasmids, viroids or the like that infect cells. Cells will belysed and the lysate usually processed in various ways familiar to thosein the art to obtain an aqueous solution of DNA or RNA, to which theseparation or isolation methods of the invention are applied. The DNA orRNA, in such a solution, will typically be found with other components,such as proteins, RNAs (in the case of DNA separation), DNAs (in thecase of RNA separation), or other types of components.

[0050] Regardless of the nature of the source of such material, thebiological target material to be isolated in the present methods isprovided in a medium comprising the biological target material and otherspecies. The biological target material must be present in the medium ina form in which it is available to adhere to the silica magneticparticles in the first step of the method. When the nucleic acidmaterial is contained inside a cell, the cell walls or cell membrane canmake the material unavailable for adhesion to the particles. Even ifsuch cells are lysed or sufficiently disrupted to cause the nucleic acidmaterial contained therein to be released into the surrounding solution,cellular debris in the solution could interfere with the adhesion of thenucleic acid material to the silica magnetic particles. Therefore, incases where the nucleic acid material to be isolated using the methodsof the present invention is contained within a cell, the cell ispreferably first processed by lysing or disrupting the cell to produce alysate, and more preferably additionally processed by clearing thelysate of cellular debris (e.g., by centrifugation or vacuum filtration)likely to interfere with adhesion of the nucleic acid material to silicamagnetic particles when provided as the medium in the methods of thepresent invention.

[0051] Any one of a number of different known methods for lysing ordisrupting cells to release nucleic acid materials contained therein aresuitable for use in producing a medium from cells for use in the presentinvention. The method chosen to release the nucleic acid material from acell will depend upon the nature of the cell containing the material.For example, in order to cause a cell with a relatively hard cell wall,such as a fungus cell or a plant cell, to release the nucleic acidmaterial contained therein one may need to use harsh treatments such aspotent proteases and mechanical shearing with a homogenizer ordisruption with sound waves using a sonicator. Contrastingly, nucleicacid material can be readily released from cells with lipid bi-layermembranes such as E. coli bacteria or animal blood cells merely bysuspending such cells in an aqueous solution and adding a detergent tothe solution.

[0052] Once the nucleic acid material is released from cells lysed ordisrupted as described above, cellular debris likely to interfere withthe adhesion of the nucleic acid material to silica magnetic particlescan be removed using a number of different known techniques orcombination of techniques. The solution of lysed or disrupted cells ispreferably centrifuged to remove particulate cell debris. Thesupernatant is then preferably further processed by adding a secondsolution to the supernatant which causes a precipitate of additionalother material to form, and then removing the precipitate from theresulting solution by centrifugation.

[0053] In a particularly preferred aspect of the present method, thenucleic acid material of interest isolated according to the method ofthe present invention is plasmid DNA initially contained in an E. colibacteria cell. The nucleic acid material is most preferably releasedfrom the bacteria cell by addition of an alkaline solution, such as asolution of sodium hydroxide, to form a lysate. The lysate is thenpreferably further treated by centrifugation to remove cell debris. Aneutralizing solution, such as an acidic buffer, is preferably added tothe resulting supernatant to form a precipitate of additionalpotentially interfering material. The precipitate thus formed ispreferably removed by centrifugation. The remaining supernatant ofcleared lysate is the medium provided in the first step of thisparticularly preferred aspect of the present method.

[0054] The medium provided in the first step of the method of thisinvention need not contain nucleic acid material released directly fromcells. The nucleic acid material can be the product of an amplificationreaction, such as amplified DNA produced using the polymerase chainreaction (PCR). The nucleic acid material can also be in the form offragments of DNA produced by digesting DNA with a restriction enzyme.The medium can also be in the form of a mixture of melted orenzymatically digested electrophoresis gel and nucleic acid material.

[0055] The silica magnetic particles provided in the second step of themethods of the present invention preferably have the capacity to form acomplex with the nucleic acid material in the medium by reversiblybinding at least 2 micrograms of nucleic acid material per milligram ofparticle. The particles provided for use in the present invention morepreferably have the capacity to reversibly bind at least 4 micrograms,and more preferably at least 8 micrograms of nucleic acid material permilligram of particle. The silica magnetic particles should preferablyhave the capacity to release at least 60% of the nucleic acid materialadhered thereto. The particles more preferably have the capacity torelease at least 70%, and most preferably at least 90% of the nucleicacid material adhered thereto. The silica magnetic particles provided inthe first step of the methods of the present invention are mostpreferably SOCM particles.

[0056] A complex of the silica magnetic particles and the biologicaltarget material is formed in the third step, preferably by exposing theparticles to the medium containing the target material under conditionsdesigned to promote the formation of the complex. The complex is morepreferably formed in a mixture of the silica magnetic particle, themedium, and a chaotropic salt.

[0057] Chaotropic salts are salts of chaotropic ions. Such salts arehighly soluble in aqueous solutions. The chaotropic ions provided bysuch salts, at sufficiently high concentration in aqueous solutions ofproteins or nucleic acids, cause proteins to unfold, nucleic acids tolose secondary structure or, in the case of double-stranded nucleicacids, melt (i.e., strand-separate). It is thought that chaotropic ionshave these effects because they disrupt hydrogen-bonding networks thatexists in liquid water and thereby make denatured proteins and nucleicacids thermodynamically more stable than their correctly folded orstructured counterparts. Chaotropic ions include guanidinium, iodide,perchlorate and trichloroacetate. Preferred in the present invention isthe guanidinium ion. Chaotropic salts include guanidine hydrochloride,guanidine thiocyanate (which is sometimes referred to as guanidineisothiocyanate), sodium iodide, sodium perchlorate, and sodiumtrichloroacetate. Preferred are the guanidinium salts, and particularlypreferred is guanidine hydrochloride.

[0058] The concentration of chaotropic ions in the mixture formed inthis practice of the present method is preferably between about 0.1 Mand 7 M, but more preferably between about 0.5 M and 5 M. Theconcentration of chaotropic ions in the mixture must be sufficientlyhigh to cause the biological target material to adhere to the silicamagnetic particles in the mixture, but not so high as to substantiallydenature, to degrade, or to cause the target material to precipitate outof the mixture. Proteins and large molecules of double-stranded DNA,such as chromosomal DNA, are stable at chaotropic salt concentrationsbetween 0.5 and 2 molar, but are known to precipitate out of solution atchaotropic salt concentrations above about 2 molar. See, e.g. U.S. Pat.No. 5,346,994 issued to Piotr Chomczynski, column 2, lines 56-63.Contrastingly, RNA and smaller molecules of DNA such as plasmid DNA,restriction or PCR fragments of chromosomal DNA, or single-stranded DNAremain undegraded and in solution at chaotropic salt concentrationsbetween 2 and 5 molar.

[0059] With any chaotropic salt used in the invention, it is desirablethat the concentration of the salt, in any of the solutions in which thesalt is employed in carrying out the invention, remain below thesolubility of the salt in the solution under all of the conditions towhich the solution is subjected in carrying out the invention.

[0060] In a practice of the present methods, the mixture formed asdescribed above is incubated until at least some of the nucleic acidmaterial is adhered to the silica magnetic particle to form a complex.This incubation step is carried out at a temperature of at least 0° C.,preferably at least 4° C., and more preferably at least 20° C., providedthat the incubation temperature is no more than 67° C. The incubationstep must be carried out at a temperature below the temperature at whichthe silica magnetic particles begin to loose their capacity toreversibly bind the nucleic acid material. The incubation step is mostpreferably carried out at about room temperature (i.e at about 25° C.).

[0061] The complex is removed from the mixture using a magnetic field.Other forms of external force in addition to the magnetic field can alsobe used to isolate the biological target substance according to themethods of the present invention after the initial removal step.Suitable additional forms of external force include, but are not limitedto, gravity filtration, vacuum filtration and centrifugation.

[0062] The external magnetic field used to remove the complex from themedium can be suitably generated in the medium using any one of a numberof different known means. For example, one can position a magnet on theouter surface of a container of a solution containing the particles,causing the particles to migrate through the solution and collect on theinner surface of the container adjacent to the magnet. The magnet canthen be held in position on the outer surface of the container such thatthe particles are held in the container by the magnetic field generatedby the magnet, while the solution is decanted out of the container anddiscarded. A second solution can then be added to the container, and themagnet removed so that the particles migrate into the second solution.Alternatively, a magnetizable probe could be inserted into the solutionand the probe magnetized, such that the particles deposit on the end ofthe probe immersed in the solution. The probe could then be removed fromthe solution, while remaining magnetized, immersed into a secondsolution, and the magnetic field discontinued permitting the particlesgo into the second solution. Commercial sources exist for magnetsdesigned to be used in both types of magnetic removal and transfertechniques described in general terms above. See, e.g. MagneSphere®Technology Magnetic Separation Stand or the PolyATract® Series 9600™Multi-Magnet, both available from Promega Corporation; MagnetightSeparation Stand (Novagen, Madison, Wis.); or Dynal Magnetic ParticleConcentrator (Dynal, Oslo, Norway).

[0063] In a preferred aspect of the methods of the present invention,the complex removed from the medium in the third step is washed at leastonce by being rinsed in a wash solution. The wash solution used in thispreferred additional step of the method preferably comprises a solutioncapable of removing contaminants from the silica magnetic particle. Thewash solution preferably comprises a salt and a solvent, preferably analcohol. The concentration of alcohol in this last preferred form of thewash solution is preferably at least 30% by volume, more preferably atleast 40% by volume, and most preferably at least 50% by volume. Thealcohol so used is preferably ethanol or isopropanol, more preferablyethanol. The salt is preferably in the form of a buffer, and mostpreferably in the form of an acetate buffer. The concentration of saltin the wash solution is sufficiently high to ensure the nucleic acidmaterial is not eluted from the silica magnetic particles during thewash step(s).

[0064] The complex is preferably washed after removal from the medium byresuspending the complex in the wash solution. The complex is preferablyremoved from the wash solution after the first wash, and washed at leastonce more, and most preferably three more times using fresh washsolution for every wash step.

[0065] Fourth, and finally, the nucleic acid material is eluted from thesilica magnetic particle by exposing the complex to an elution solution.The elution solution is preferably an aqueous solution of low ionicstrength, more preferably water or a low ionic strength buffer at abouta pH at which the nucleic acid material is stable and substantiallyintact. Any aqueous solution with an ionic strength at or lower than TEbuffer (i.e. 10 mM Tris-HCl, 1 mM ethylenediamine-tetraacetic acid(EDTA), pH 8.0) is suitable for use in the elution steps of the presentmethods, but the elution solution is preferable buffered to a pH betweenabout 6.5 and 8.5, and more preferably buffered to a pH between about7.0 and 8.0. TE Buffer and distilled or deionized water are particularlypreferred elution solutions for use in the present invention. The lowionic strength of the preferred forms of the elution solution describedabove ensures the nucleic acid material is released from the particle.Other elution solutions suitable for use in the methods of thisinvention will be readily apparent to one skilled in this art.

[0066] The nucleic acid material eluted from the complex in the elutionstep of the method is preferably separated from the silica magneticparticles and complexes remaining in the elution mixture by externalforce, such as centrifugation or a magnetic field, but more preferablyusing centrifugation. Centrifugation is preferred because it can resultin the removal of particles or particle fragments which are too small orwhich are not sufficiently magnetically responsive to be removed byusing a magnetic field.

[0067] The nucleic acid material eluted using the method of the presentinvention is suitable, without further isolation, for analysis orfurther processing by molecular biological procedures. The elutednucleic acid can be analyzed by, for example, sequencing, restrictionanalysis, or nucleic acid probe hybridization. Thus, the methods of theinvention can be applied as part of methods, based on analysis of DNA orRNA, for, among other things, diagnosing diseases; identifyingpathogens; testing foods, cosmetics, blood or blood products, or otherproducts for contamination by pathogens; forensic testing; paternitytesting; and sex identification of fetuses or embryos.

[0068] The eluted DNA or RNA provided by the method of the invention canbe processed by any of various exonucleases and endonucleases thatcatalyze reactions with DNA or RNA, respectively, and, in the case ofDNA, can be digested with restriction enzymes, which cut at restrictionsites present in the DNA. Restriction fragments from the eluted DNA canbe ligated into vectors and transformed into suitable hosts for cloningor expression. Segments of the eluted DNA or RNA can be amplified by anyof the various methods known in the art for amplifying target nucleicacid segments. If eluted DNA is a plasmid or another type ofautonomously replicating DNA, it can be transformed into a suitable hostfor cloning or for expression of genes on the DNA which are capable ofbeing expressed in the transformed host. Plasmid DNAs isolated bymethods of the present invention have been found to be more efficientlytransfected into eukaryotic cells than those isolated by the prior artmethod, wherein diatomaceous earth is employed in place of the silicagel in the methods of the invention of this application.

[0069] The following, non-limiting examples teach various embodiments ofthe invention. In the examples, and elsewhere in the specification andclaims, volumes and concentrations are at room temperature unlessspecified otherwise. Only the most preferred form of the magnetic silicaparticles was used in used in each of the examples below, i.e. SOCMparticles. However, one skilled in the art of the present invention willbe able to use the teachings of the present disclosure to select and useforms of the magnetic silica particles other than the SOCM particleswhose use is illustrated in the aspects of the methods of the presentinvention demonstrated in the Examples below.

[0070] The same batch of SOCM particles was used to produce the assayresults presented in Examples 1 and 6 below, while a second batch ofSOCM particles was used to generate the results presented in Examples2-4 and 7. However, both batches of SOCM particles were found to produceacceptable results when tested as described below. The first batch ofSOCM particles, i.e. the particles used in Examples 1 and 6, were foundto have the following physical characteristics: surface area of 55 m²/g,pore volume of 0.181 ml/g for particles of <600 Å diameter, pore volumeof 0.163 ml/g for particles of >600 Å diameter, median particle size of5.3 μm, and iron leach of 2.8 ppm when assayed as described herein aboveusing ICP. The other batch of SOCM particles used in the Examples belowwere found to have the following characteristics: surface area of 49m²/g, pore volume of 0.160 ml/g (<600 Å diameter), pore volume of 0.163ml/g (>600 Å diameter), median particle size of 5.5 μm, and iron leachof 2.0 ppm.

EXAMPLE 1 Assay of Binding Capacity and Elution Efficiency of SilicaMagnetic Particles for Plasmid DNA

[0071] The binding capacity of the SOCM form of silica magneticparticles and of magnetic controlled pore glass (CPG) particles wasdetermined by titrating increasing amounts of plasmid against a constantamount of particles in a final 600 μl volume of 4.16M guanidinehydrochloride (GHCI). The magnetic CPG particles used were 5 μM magneticglass particles with a 500 Å average pore size, obtained from CPG Inc.,Lincoln Park, N.J., U.S.A., Part Number MCPGO510).

[0072] In the present example, a 140mg of magnetic silica was suspendedin 10 ml of deionized water (DI H₂O) and then washed 3 times with 10 mlof SM GHCI before being suspended at a final concentration 14 mg/ml inthe same solution. A binding mixture was formed by adding increasingvolumes pGEM® 3zf(+) plasmid DNA from Promega Corporation (CatalogNumber P2271) in DI H₂O at a concentration of 1.0 micrograms (μg) perμl, corresponding to 5 μg, 10 μg, 20 μg, 40 μg, 60 μg and 80 μg of DNA,to 500 μl of the particles and brought to a final volume of 600 μl bythe addition of DI H₂O. The plasmid/particle binding mixture was thenincubated for 2-3 minutes at room temperature.

[0073] The amount of plasmid bound to the magnetic silica was determinedby subtracting the amount plasmid DNA remaining in solution from thetotal amount of plasmid added to the particles in each sample, asfollows. The liquid fraction of the assay mixture was separated from themagnetic silica by centrifugation at 14,000× g for 20 seconds. Theamount of plasmid DNA remaining in the supernatant was determined bymonitoring the absorbency of the solution at 260 nm. One absorbency unitat 260 nm is equivalent to a plasmid DNA concentration of 50 μg/ml.

[0074] The silica magnetic particles remaining in the binding mixturewere then separated from the mixture and washed as follows. A magnet waspositioned outside the container holding the binding mixture but closeto one side of the container, causing the silica magnetic particles inthe mixture to deposit on the side of the container closest to themagnet. The magnet was then maintained in its position on the side ofthe container while the mixture was decanted out of the container,leaving the substantially all the silica magnetic particles in thecontainer. The remaining silica magnetic particles where then washed 4times with 1 ml of a wash solution of 80 mM KOAc and 10 μM EDTAcontaining 55% EtOH, removing the magnet from the side of the containerduring each washing step and positioning the magnet, once again, on aside of the container to ensure the particles remain in the containerwhile the wash solution is decanted following each washing step. Theparticles remaining in the container after the last wash step were thenair dried for 3-5 minutes.

[0075] Finally, the plasmid DNA was eluted from the silica magneticparticles by adding 1 ml of DI water at room temperature. The particleswere removed from the resulting isolated plasmid DNA solution bycentrifugation. The amount of plasmid DNA eluted was then determined bymeasuring the absorbency of the solution at 260 nm.

[0076] The overall efficiency of the plasmid isolation process wasdetermined as the percent of DNA recovered in the final elution comparedto the amount of DNA incubated with the particle. The binding capacitywas determined at the point where the overall efficiency dropped to 90%.

[0077] The results of the binding assay described above are presented inFIG. 1, and together with the elution results in FIG. 3. The DNA bindingcapacity results obtained with the magnetic silica (Δ) and magnetic CPG(+) particles are shown separately in FIG. 1. The results show that asincreasing amounts of plasmid DNA was added to the magnetic silicaparticles, the particles continued to bind increasing amounts of DNA,binding as much as 90 μg per 130 μg of plasmid added. Contrastingly, themagnetic CPG particles failed to bind more than 40 μ/g of plasmid DNAeven when 130 μg of plasmid DNA was added. The total binding capacity ofthe silica magnetic particles was 8 μg of plasmid per mg of particle.This is significantly higher than the binding capacity of the magneticCPG particles, and at least 4-fold higher than the binding capacity ofthe 10 μM silica bead used in Promega Corporation's Wizard™ Plus PlasmidDNA Purification Systems.

[0078] The results of the elution assay described above are presented inFIG. 2, and together with the elution results in FIG. 3. The resultsshow that greater than 90% of the plasmid DNA bound to the silicamagnetic particles in this example was eluted from the particles, whileless than 60% of the plasmid DNA bound to the CPG particles was elutedtherefrom.

[0079] The results displayed in FIGS. 1-3 clearly demonstrate that thesilica magnetic particles assayed herein exhibit excellent binding andelution characteristics.

EXAMPLE 2 Assay of Binding Capacity and Elution Efficiency of SilicaMagnetic Particles for DNA Fragments

[0080] Purified native lambda DNA from Promega Corporation (CatalogNumber D150) was digested with Hind III restriction enzyme, an enzymewhich cuts native lambda DNA into 8 fragments ranging in size from23,000 bp to 125 bp. This Hind III digested lambda DNA is referred tohereinafter as “λ Hind III digest.”

[0081] The magnetic silica was prepared as described previously andresuspended in 5M GHCI at a concentration of 14 mg/ml. One ml of theresuspended particle solution was incubated with 80 μl of the λ Hind IIIdigest (0.44 μg/μl) for 2-3 minutes at room temperature. The amount ofDNA bound to the magnetic silica was determined by subtracting the DNAremaining in solution from the total amount of DNA added to theparticles after separation of the liquid and solid phases bycentrifugation at 14,000× g for 20 seconds. DNA concentrations weredetermined by absorbency measurement at 260 nm. One absorbency unit at260 nm is equivalent to a DNA concentration of 50 μg/ml.

[0082] The silica magnetic particles remaining in the binding mixturewere then separated from the mixture and washed as follows. A magnet waspositioned outside the container holding the binding mixture but closeto one side of the container, causing the silica magnetic particles inthe mixture to deposit on the side of the container closest to themagnet. The magnet was then maintained in its position on the side ofthe container while the mixture was decanted out of the container,leaving the substantially all the silica magnetic particles in thecontainer. The remaining silica magnetic particles where then washed 4times with 1 ml of a wash solution of 80 mM KOAc and 10 μM EDTAcontaining 55% EtOH, removing the magnet from the side of the containerduring each washing step and positioning the magnet, once again, on aside of the container to ensure the particles remain in the containerwhile the wash solution is decanted following each washing step. Theparticles remaining in the container after the last wash step were thenair dried for 3-5 minutes.

[0083] Finally, the λ Hind III digest was eluted by adding 200 μ/l of DIwater at room temperature. The particles were removed from the resultingisolated A digest solution by centrifugation. The amount of λ Hind IIIdigest DNA eluted was then determined by measuring the absorbency of thesolution at 260 nm.

[0084] Similar silica magnetic particle binding and elution assays wereperformed using φ174 DNA digested with Hae III restriction enzyme, adigestion reaction which produces 10 DNA fragments ranging from 1353 bpto 72 bp in size. The data for these experiments are summarized in Table1, below. TABLE 1 DNA DNA % DNA Type DNA Added Bound Eluted* Recovery**λ HindIII Digest 35.0 μg 33.6 μg 28.4 μg 81.0% φX174 HaeIII 40.0 μg 39.4μg 33.7 μg 84.2% Digest

EXAMPLE 3 Electrophoresis of DNA Fragments after Elution from SilicaMagnetic Particles

[0085] In order to determine whether the silica magnetic particles boundor released DNA fragments of different molecular weights at differentweights, the DNA fragments bound to and eluted from the silica magneticparticles in Example 2 were assayed using electrophoresis as follows.Samples of λ Hind III digest eluted from two different samples of silicamagnetic particles were loaded and fractionated on an agarose gel alongwith a control sample of untreated DNA digest. Samples of bound andeluted φX174 Hae III digest were also fractionated on an agarose gelalong with a control sample of untreated DNA digest. The resulting gelsof fractionated DNA were then stained with a fluorescent dye capable ofstaining DNA, and the stained gels analyzed using a Molecular DymanicsFluoroimager. The fluorescent intensity of the eluted DNA fragments fromeach of the restriction enzyme digests were compared to the controldigests prior to capture and elution on magnetic silica.

[0086]FIGS. 4 and 5 show the visual image generated by the fluorometerfrom the fluorescent stained agarose gel of fractionated captured andeluted DNA fragments produced as described above. FIG. 4 shows 2 μg ofλHindIII digest electrophoesed on 1% agarose gel. FIG. 5 shows 5 μg ofφX174 Hae III digest electrophoesed on 3% agarose gel. In both panels,sample 1 is the control of untreated DNA digest, while samples 2 and 3are samples of digest DNA bound to and eluted from two different samplesof silica magnetic particles.

[0087] No substantial difference in relative band intensity orbackground was noted between the control and samples from either set ofdigest samples analyzed herein, indicating the silica magnetic particlesassayed herein do not selectively bind or release DNA fragmentsaccording to molecular weight.

EXAMPLE 4 Isolation of Plasmid DNA from Bacterial Cultures Using SilicaMagnetic Particles and Magnetic Force

[0088] Some of the resuspended silica magnetic particles prepared inExample 1 were used to isolate pGEM®-3zf(+) plasmid DNA from a cultureof DH5α E. coli bacteria transformed with either form of plasmid DNA.The following solutions were used in the isolation procedure:

[0089] 1. Cell Resuspension Solution:

[0090] 50 mM Tris-HCl, pH 7.5

[0091] 10 mM EDTA

[0092] 100 μg/ml DNase-free ribonuclease A (RNase A)

[0093] 2. Column Wash Solution:

[0094] Prepared by making an aqueous buffer consisting of either

[0095] 200 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, pH 7.5, or

[0096] 190 mM KOAc, 20 mM Tris-HCl, 0.1 mM EDTA, pH 7.5,

[0097] and by diluting the aqueous buffer 1:1.4 with 95% ethanol (EtOH).

[0098] 3. TE Buffer:

[0099] 10 mM Tris-HCl, pH 7.5

[0100] 1 mM EDTA

[0101] 4. Neutralization Solution:

[0102] 1.32M KOAc (potassium acetate), pH 4.8

[0103] 5. Cell Lysis Solution:

[0104] 0.2M NaOH

[0105] 1% SDS (sodium dodecyl sulfate)

[0106] The bacteria culture was treated to produce a cleared lysate, byfollowing the steps described briefly below:

[0107] 1. The cells from 1 to 3ml of bacteria culture were harvested bycentrifuging the culture for 1-2 minutes at top speed in amicrocentrifuge. The harvested cells were resuspended in 200 μl of CellResuspension Solution, and transferred to a microcentrifuge tube. Theresulting solution of resuspended cells was cloudy.

[0108] 2. 200 μl of Cell Lysis Solution was then added to the solutionof resuspended cells and mixed by inversion until the solution becamerelatively clear, indicating the resuspended cells had lysed.

[0109] 3. 200 μl of Neutralization Solution was added to the lysatesolution, and mixed by inversion. The lysate became cloudy after theNeutralization Solution was added.

[0110] 4. The solution was then spun in a microcentrifuge at top speedfor 5 minutes to clear the lysate.

[0111] 5. The resulting supernatant of cleared lysate was transferred toa new microcentrifuge tube.

[0112] Plasmid DNA was then isolated from the cleared lysate using thesilica magnetic particles suspended in a solution of guanidinehydrochloride prepared in Example 1. Essentially the same procedure wasused to isolate the plasmid DNA using the particles and magnetic force,as was used in the plasmid binding assay described in Example 2.However, the present isolation procedure was initiated by adding 1 ml ofthe suspended silica magnetic particles to the cleared lysate producedfrom step 5, immediately above, rather than beginning the procedure byadding 500 μl of suspended particles to 5 to 80 μg of purified plasmidDNA. The volumes of each solution added to the magnetic silica particlesat each subsequent step of the present isolation procedure followed wereadjusted proportionately to account for the larger starting volume.

[0113] The resulting isolated plasmid DNA was assayed qualitativelyusing gel electrophoresis, and quantitatively using a spectrophotometer.the gel assay results showed a high percentage of intact, supercoiledplasmid DNA present in the sample. The optical density measurementsaccurately reflected DNA yield, as evidenced by absorbance ratios (ex.260/250 nm and 260/280 nm) in the expected range for DNA.

EXAMPLE 5 Isolation of Plasmid DNA from Bacterial Cultures Using SilicaMagnetic Particles and Vacuum Filtration

[0114] The same procedure is used to produce a cleared lysate of aculture of E. coli bacteria transformed with plasmid DNA, such as thecleared lysate production procedure used in Example 4. The plasmid isthen isolated from the resulting cleared lysate using the suspension ofsilica magnetic particles of Example 1, but using vacuum filtrationrather than magnetic force to separate the particles from the bindingmixture once the plasmid DNA has adhered to the particles. Vacuumfiltration is also used to remove the wash solution from the particlesin the washing steps of the isolation procedure.

EXAMPLE 6 Illustrating Binding of RNA

[0115] Using magnetic silica at 14 mg/ml in 4M Guanidine Thiocynate, 700μl of resuspended silica magnetic particles prepared as in Example 1were added to 30 μl of Promega RNA Markers, catalog # 1550, labeled with³²P (app. 200,000 cpms), and 5 μl of a 1 mg/ml solution of cold (i.e.unlabeled) Promega RNA Markers part #G3191 in a container.

[0116] The resulting mixture was incubated for 5 minutes at roomtemperature, after which the particles were captured, using magneticforce to draw the particles to one side of the container while thesupernatant was decanted into a second container.

[0117] The supernant collected in the second container was saved andcounted.

[0118] The captured particles in the first container were then washedthree times with Column Wash Solution, prepared as described in Example4, above. The particles were captured after each wash step, and the washsolution decanted. Each decanted wash solution was saved and counted.The sum of the supernant counts and the wash void counts was used todetermine the total unbound CPMs.

[0119] After the third wash step, the RNA was eluted from the capturedand washed particles by resuspending the particles in 250 μl of Nanopurewater heated to 37° C., and then using magnetic force to hold theparticles on the side of the container while the eluent was decanted andcollected. 100 μl of the eluent was then counted.

[0120] The remaining particles were resuspended in 500 μl of Nanopurewater and then counted to determine the amount of uneluted CPMsremaining.

[0121] The above analysis was run in duplicate. The results shown inFIG. 6 reflect the counts averaged for each set of duplicates and countscollected in each experiment. FIG. 6 shows that of 200,000 CPMs of RNAexposed to the magnetic silica particles in this assay, an average of125,000 CPMs became bound to the particles, and about 100,000 of theCPMs bound to the particles was released and eluted from the particlesin the final elution step.

[0122] These RNA binding and elution assay results are comparable to theDNA binding and elution results described in Example 2, above. Thepresent assay shows the potential application of the magnetic silicaparticles according to the methods of the present invention to isolateRNA.

EXAMPLE 7 Analysis of Iron Leach from Silica Magnetic Particles

[0123] Silica magnetic particles, such as those used in the Examplesabove, were screened for their tendency to leach iron or other materialslikely to interfere with the quantitative analysis of nucleic acidmaterials, by producing an absorbance peak at or around 260 nm whensolutions exposed to the particles are analyzed with aspectrophotometer.

[0124] The silica magnetic particles were analyzed as follows. 140 mg ofsilica magnetic particles were resuspended in 10 ml of DI water andvortexed briefly. The particles were exposed to a magnetic field for 1minute, by placing a magnet against the outside of the container holdingthe particle/water mixture. Particles in the mixture collected on theside of the container closest to the magnet surface, and were heldagainst the side of the container by the magnet while the supernatantwas decanted out of the container. The magnet was then removed, and theparticles remaining in the container were resuspended in another 10 mlof DI water. The collection, decanting, and resuspension steps wererepeated three times.

[0125] After the third such step, the resuspended particles weresequentially washed twice with 10 ml each of 7M guanidine hydrochloride,pH 5.9, twice with 10 ml of DI water, and twice with 10 m. of 50 mM EDTA(pH 8.0). The supernatants from each of these washes were scanned from230 nm to 300 nm using a Hewlett Packard Diode array spectrophotometerblanked against each of the control solutions.

[0126] No absorbance above background at 260 nm was observed in any ofthe wash solutions obtained by assaying the silica magnetic particlesused in the Examples above.

We claim:
 1. A method for isolating a biological target material fromother material in a medium by: a. providing a medium including thebiological target material; providing silica magnetic particles capableof reversibly binding the biological target material; b. forming acomplex of the silica magnetic particles and the biological targetmaterial by combining the silica magnetic particles and the medium; c.removing the complex from the medium by application of an externalmagnetic field; and d. separating the biological target material fromthe complex by eluting the biological target material, whereby theisolated biological target material is obtained.
 2. A method ofisolating a biological target material according to claim 1, wherein thebiological material isolated according to the method consists of anucleic acid.
 3. A method of isolating a biological target materialaccording to claim 1, wherein the silica magnetic particles provided instep (b) are capable of reversibly binding at least 2 micrograms ofbiological target material per milligram of particle.
 4. A method ofisolating a biological target material according to claim 3, wherein thesilica magnetic particles provided in step (b) of the method aresiliceous-oxide coated magnetic particles.
 5. A method of isolating abiological target material according to claim 1, wherein at least 60% ofthe biological target material in the complex is eluted from theparticles in step (d).
 6. A method of isolating a biological targetmaterial according to claim 1, wherein the biological target materialeluted from the complex in step (d) contains no more than 50 parts permillion of transition metal contaminants.
 7. A method of isolating abiological target material from other materials in a medium comprisingthe steps of: a) providing a medium containing the biological targetmaterial; b) providing a silica magnetic particle with the capacity toreversibly bind at least 2 micrograms of biological target material permilligram of particle; c) forming a mixture comprising the medium andthe silica magnetic particle; d) adhering the biological target materialto the silica magnetic particle in the mixture; e) removing the silicamagnetic particle with the biological target material adhered theretofrom the mixture by application of an external magnetic field; and f)eluting at least 60% of the biological target material from the silicamagnetic particle by exposing the particle to an elution solution.
 8. Amethod of isolating a biological target material according to claim 7,wherein the biological material isolated according to the methodconsists of a nucleic acid material.
 9. A method of isolating abiological target material according to claim 8, wherein the nucleicacid biological target material isolated according to the methodconsists of a plasmid DNA material.
 10. A method of isolating abiological target material according to claim 8, wherein the nucleicacid biological target material isolated consists of DNA fragmentmaterial.
 11. A method of isolating a biological target materialaccording to claim 7, wherein the silica magnetic particles provided instep (b) of the method are siliceous oxide-coated magnetic particles.12. A method of isolating a biological target material according toclaim 7, wherein the mixture formed in step (c) comprises the medium,the silica magnetic particle, and a chaotropic salt, wherein thechaotropic salt concentration is sufficiently high to cause thebiological target material to adhere to the silica magnetic particle instep (d).
 13. A method of isolating a biological target materialaccording to claim 12, wherein the chaotropic salt in the mixture formedin step (c) consists of a guanidinium chaotropic salt consisting ofguanidine hydrochloride or guanidine thiocyanate.
 14. A method ofisolating a biological target material according to claim 12, whereinthe concentration of chaotropic salt in the mixture formed in step (c)is at least 2 molar.
 15. A method of isolating a biological targetmaterial according to claim 7, wherein the biological target material isadhered to the silica magnetic particle in step (d) by incubating themixture.
 16. A method of isolating a biological target materialaccording to claim 15, wherein the biological target material is adheredto the silica magnetic particle in step (d) by incubating the mixture atroom temperature for at least 30 seconds.
 17. A method of isolating abiological target material according to claim 7, further comprising astep of washing the silica magnetic particle after removal from themedium, before eluting the biological target material from the particle.18. A method of isolating a biological target material of claim 17,wherein the washing step is done using a wash solution comprising analcohol and a salt.
 19. A method of isolating a biological targetmaterial according to claim 18, wherein the washing step is done using awash solution comprising at least 30% alcohol by volume and a buffer.20. A method of isolating a biological target material according toclaim 7, wherein the biological target material is eluted from thesilica magnetic particle in step (f) using water or an elution solutionwith a low ionic strength.
 21. A method of isolating biological targetmaterial according to claim 7, wherein the biological target materialeluted from the silica magnetic particle in step (f) is substantiallyfree of macromolecular or metal contaminants.
 22. A method of isolatinga plasmid DNA material from other materials in a medium comprising thesteps of: a) providing a medium containing the plasmid DNA; b) providinga siliceous oxide-coated magnetic particle with the capacity toreversibly bind at least 2 micrograms of biological target material permilligram of particle; c) forming a mixture comprising the medium, thesiliceous oxide-coated magnetic particle, and a chaotropic salt, whereinthe chaotropic salt concentration in the mixture is sufficiently high tocause the plasmid DNA to adhere to the particle; d) incubating themixture at about room temperature until at least some of the biologicaltarget material is adhered to the siliceous oxide-coated magneticparticle; e) removing the siliceous oxide-coated magnetic particle fromthe mixture using an external magnetic force; and f) eluting at least60% of the plasmid DNA adhered to the siliceous oxide-coated magneticparticle by exposing the particle to an elution solution.
 23. A methodof isolating a plasmid DNA material according to claim 22, wherein thechaotropic salt in the mixture formed in step (c) is a guanidiniumchaotropic salt consisting of guanidine hydrochloride or guanidinethiocyanate.
 24. A method of isolating a plasmid DNA material accordingto claim 22, wherein the concentration of chaotropic salt in the mixtureformed in step (c) is between about 0.1 M and 7 M.
 25. A method ofisolating a plasmid DNA material according to claim 22, furthercomprising a step of washing the siliceous oxide-coated magneticparticle after removal from the medium, before eluting the plasmid DNAmaterial from the particle.
 26. A method of isolating a plasmid DNAmaterial according to claim 25, wherein the washing step is done using awash solution comprising an alcohol and a salt.
 27. A method ofisolating a plasmid DNA material according to claim 25, wherein thewashing step is done using a wash solution comprising at least 30%alcohol by volume and a buffer.
 28. A method of isolating a plasmid DNAmaterial according to claim 22, wherein the plasmid DNA eluted from thesilica magnetic particle in step (f) is substantially free ofmacromolecular or metal contaminants likely to interfere with furtherprocessing or analysis.
 29. A kit for isolating a biological targetmaterial from a medium, the kit comprising: an aliquot of siliceousoxide-coated magnetic particles suspended in an aqueous solution in afirst container, wherein the particles have the capacity to reversiblybind at least 2 micrograms of the biological target material permilligram of particle.
 30. A kit for isolating a biological targetmaterial according to claim 29, further comprising: a chaotropic salt ina second container; and a wash solution in a third container.